Transfection with magnetic nanoparticles and ultrasound

ABSTRACT

The invention includes a magnetic nanoparticle molecular delivery vehicle to be used for transfection and delivery of therapeutic molecules across cell membranes and to specific sites in the body, using magnetic forces and ultrasound.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is filed under 37 CFR 1.53(b) as a continuation-in-partapplication. This application claims priority under 35 USC §120 of U.S.patent application 13/127,259 filed on May 3, 2011, and which is theU.S. National Stage of International Application No. PCT/CA2009/001629,filed Nov. 9, 2009, which designates the U.S., published in English, andwhich claims the benefit of U.S. Provisional Application No. 61/112,451,filed Nov. 7, 2008, the specifications of which are hereby incorporatedby reference.

FIELD OF THE INVENTION

This invention relates to a nanostructured molecular delivery vehiclefor delivering molecules to a selected site, and a method fortransporting the molecular delivery vehicle across a biological membraneby applying a magnetic force and ultrasound.

BACKGROUND OF THE INVENTION

Transfection is the introduction of foreign genetic material intoeukaryotic cells using a vector as a means of transfer. The termtransfection is most often used in reference to mammalian cells, whilethe term transformation is preferred to describe DNA transfer inbacteria and non-animal eukaryotic cells such as fungi, algae andplants.

Existing methods of transfection must overcome problems with thepermeability of the cell membrane and the solubility of the transfectedparticle.

Drug delivery often involves transportation of the drug across cellmembranes. The most basic method in vivo method is to introduce the druginto the blood stream by oral or intravenous methods and then hope it isabsorbed by the correct cells. This non-discriminatory techniquerequires relatively large doses of the drug and simply does not work forsome molecules such as DNA, which is used in gene therapy.

Existing methods to transfect material into a cell can be grouped intotwo categories: viral and non-viral. The utilization of viruses totransfect material into a cell has been shown to be extremely efficient;however, the possibility of a immune response to viruses and theinsertion of mutagens into the body have proven to be serious concerns,especially in clinical trials. Non-viral drug delivery methods includenaked DNA injection and electroporation. Unfortunately, naked plasmidDNA injection has shown to have a relatively low efficiency of genedelivery, while following electroporation tissue damage caused by theelectric pulses has been observed.

Microinjection is a mechanical technique that utilizes a precision toolto place the molecule directly into the cell. This works excellently forDNA, however it is impractical in many situations as it can only beapplied to one cell at a time.

A gene gun is a mechanical device that fires a particle bonded to thebio-molecule into the cell. These particles are relatively large andoften damage cells. They also require large doses to be effective.

Electroporation is a physical method, which creates pores in the cellmembrane by applying an electric shock to the cell. These pores allowthe increased diffusion of materials into the cell. This increasedpermeability allows for easier transfection.

Sonoporation is similar to electroporation except it uses ultrasound tostimulate the cell membrane. The ultrasound also creates turbulence inthe fluid surrounding the cell, which increases the rate of diffusionacross the membrane.

Calcium phosphate transfection is a chemical method, which is verycheap. It uses calcium phosphate bonded to DNA. This molecule in somecases is able to transfect cells; however, this method is oftenineffective and limited.

Viral delivery is a very effective method because viruses naturally area carrier of genetic information and are very adept at entering cells.This makes them an obvious choice to help deliver DNA molecules intocells. However, the use of viral vectors is sometimes undesirablebecause of their immunogenicity and their potential mutagenicity.Furthermore, viral delivery is non-specific and can trigger side effectsin the host.

Yet another method uses magnetic force and a molecular delivery vehicleto cross the cell membrane. A version of this method is described inUnited States Patent Application 2007/0231908 A1, and requires that themolecular delivery vehicle be oriented before it penetrates thebiological membrane.

For most of the above methods, the effectiveness is extremely variabledepending on the cell type being transfected. Some cells are known to beharder to transfect then others and these are usually the cells thathold the greatest reward.

Therefore, there is a need in the art for methods of transportingbiomolecules and other molecules of interest into cells which mitigatethe difficulties of the prior art.

SUMMARY OF THE INVENTION

The present invention provides for transfection of cells usingnanoparticles and magnetic forces to direct the nanoparticles through acell wall or membrane. In one embodiment, the nanoparticle is directedthrough a cell membrane, a nuclear membrane, or a cell membrane in vivosuch as the blood-brain barrier. In one embodiment, the inventionfurther comprises the use of ultrasound to increase the permeability ofthe biological membranes. This results in greater efficiency ormolecular delivery or transfection.

This invention comprises the following aspects (a) a method of creatingnanoparticles, which are nontoxic, magnetic, and bondable to biologicalmolecules or other molecules of interest; (b) a method of bonding suchmolecules to this nanoparticle; and (c) a system to force thesenanoparticles through a membrane using a magnetic field. In oneembodiment, ultrasound in the form of low-intensity pulsed ultrasound(LIPUS) is used increase the permeability of the membrane.

In one aspect, the invention comprises a method of delivering a moleculeacross a cell membrane using a delivery vehicle comprising a magneticnanoparticle, the method comprising the steps of:

(a) fixing the molecule to the nanoparticle;

(b) positioning the nanoparticle in the immediate vicinity of the cellmembrane;

(c) subjecting the nanoparticle and cell membrane magnetic field; and

(d) simultaneously subjecting the nanoparticle and cell membrane toultrasound.

The nanoparticle comprises bonding sites so that the molecule can beattached to this nanoparticle. The number of bonding sites is variableas is the spacing between bonding sites. In addition, the type of bondmay be covalent, ionic or another bond which is capable of fixing themolecule to the nanoparticle. In one embodiment, the molecule maycomprise a genetic material such as DNA or RNA, proteins, or any otherbiological molecule.

The nanoparticle may comprise nanotubes, such as carbon nanotubes, orsingle-walled carbon nanotubes. In one embodiment, the nanoparticles maybe biodegradable or biocompatible, and may comprise silica. Thenanoparticles may display low or no toxicity to cells in vivo or invitro.

On a macroscopic scale, this magnetic force can be used to control themolecular delivery vehicles to move to certain parts of a body. On amicroscopic to nanoscale level, this force can be used to force themolecular delivery vehicles through a biological membrane. If necessaryor desired, the molecular delivery vehicle can be further transportedinto the nucleus of the cell by moving it with a magnetic force.

This membrane may be the cell wall or the wall of the nucleus inside thecell, or another biological membrane such as the mitochondrion's doublemembrane. This membrane could also be the blood-brain barrier. Thus,this invention may allow for the transportation of molecules into thecentral nervous system.

Thus using this method, a bio-molecule can be delivered to a specifictarget.

In one embodiment, the invention comprises a molecular delivery vehiclewhich comprises a nanostructure which is magnetic and has bonding sitesso that a bio-molecule can be attached to this molecular deliveryvehicle. The number of bonding sites is variable as is the spacingbetween bonding sites. In addition, the type of bond may be covalent,ionic or another bond which is capable of holding the biomolecule.

Using this magnetic force the magnetic nanoparticle can be controlled innumerous ways. In one embodiment, the delivery vehicles can be collectedin one location using a magnetic force that attracts to that location,such as an organ or specific tissue in vivo.

In one aspect, the invention comprises a method for using the moleculardelivery system to deliver molecules into cells or transfect such cellsin vitro or in vivo. In vitro cells may be supported on solid or liquidmedia.

In one embodiment, the cell membrane may be from a cell chosen from amammalian cell and a plant cell. The mammalian cell may be chosen from anormal cell or a cancer cell.

The plant cell may further comprise a cell wall.

The plant cell may be chosen from a canola cell or a carrot cell.

The cancer cell may be chosen from a MCF-7 cell, a HeLa cell, a KG-1cell, a breast cancer cell, a cervic cancer cell, and a human acuteleukemia cell.

The magnetic nanoparticle may be chosen from a magnetic goldnanoparticle (mGNP), a magnetic single wall carbon nanotube (mSWCNT), orcombinations thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

In order that the above-recited and other features and advantages of thepresent invention will be readily understood, a more particulardescription of the invention is given. Specific examples thereof aredetailed, the result of which are illustrated in the appended figures.Any example is only a single embodiment of the invention, and is not tobe considered in any way the limit of its scope. In the accompanyingfigures:

FIG. 1A is a sketch of a magnetic single walled nanotube and FIG. 1B isa sketch of a spherical magnetic nanoparticle after it has beenfunctionalized.

FIG. 2 shows the delivery vehicle being forced though the cell membrane.The arrows depict the magnetic field. In this depiction the carbonnanotube is being used for the delivery.

FIG. 3 depicts the use of a magnet to collect the nanoparticles at acertain location in the body. In this case the particles are beingcollected at the top of the patients left arm.

FIGS. 4A, 4B, 4C, and 4D show schematic processes for functionalizing asingle-walled nanotube.

FIGS. 5A and 5B show XPS and IR spectra for carboxylated SWNTs.

FIGS. 6A and 6B show IR and UV-vis spectra for FITC labelled SWNT. Thevertical axis A shows absorption.

FIG. 7A shows a confocal microscopy image showing control cells. FIG. 7Bshows cells a confocal microscopy image showing cells with FITC labellednanoparticles in the cytoplasm. FIGS. 7C and 7D show confocal microscopyof MCF-7 control cells and cells transfected with nanoparticles boundwith GFP plasmid.

FIG. 8A show distribution of FITC labelled nanoparticles in controlMCF-7 cells and FIG. 8B shows distribution in MCF-7 cells exposed FITClabelled magnetic nanoparticles and a magnetic field.

FIG. 9 shows a graph of percentage uptake by MCF-7 cells.

FIGS. 10A, 10B, and 10C show FITC labelled nanoparticles delivered intohematopoietic stem cells in a control, after 3 hours and after 6 hours,respectively.

FIG. 11 shows a graph demonstrating viability of MCF-7 cells after FITClabelled nanoparticle uptake compared to control cells.

FIG. 12A shows FACS results for Negative control sample contained no GFPplasmid, no Definity, and was not sonicated. FACS results: Marker: MI, %Gated: 0.16. Extremely high cell viability is observed. FIG. 12B showsFACS results for Positive control sample contained 2 μg of GFP plasmid,no Definity, the lipofection agent PEI, and was not sonicated. FACSresults: Marker: MI, % Gated: 33.12%. Very low cell viability isobserved.

FIG. 13 shows FACS results FACS results for sample sonicated at 0.5W/cm², with a 20% duty cycle for 60 seconds. DNA plasmid concentrationwas varied. FIG. 13A-DNA plasmid concentration: 2 μg/mL, marker: MI, %Gated: 16.20. FIG. 13B-DNA plasmid concentration: 15 μg/mL, marker: MI,% Gated: 26.93. FIG. 13C-DNA plasmid concentration: 30 μg/mL, marker:MI, % Gated: 32.51. A high amount of cell viability is seen in allcases.

FIG. 14 shows FACS result for sample sonicated at 0.3 W/cm², with a 100%duty cycle for 60 seconds. DNA plasmid concentration was 30 μg/mL. FACSresults: marker: MI, % Gated: 14.67. Cell viability is observed to havedecreased.

FIG. 15 FACS result for sample sonicated at 0.5 W/cm², with a 100% dutycycle for 60 seconds. DNA plasmid concentration was 30 μg/mL. FACSresults: marker: MI, % Gated: 32.12. Cell viability is observed to below.

FIG. 16 shows a picture of a biocompatible silica nanotube.

FIG. 17 shows a graph of IR spectra of Si-NT which has beencarboxylated.

FIG. 18 shows HeLa cells which have been transfected with Si-NT-GFPplasmid, compared with a control.

FIG. 19 shows a graph demonstrating low toxicity of the Si-NT after 48and 72 hours of incubation.

FIG. 20 shows the mSWCNT characteristics and synthetic process ofmSWCNT-FITC. A: AFM image of mSWCNT; B: AFM height analysis (about 30nm) of mSWCNTs in image A; C: TEM image of mSWCNT: D: mSWCNT-FITCcovalent linking process

FIG. 21 shows FITC delivery efficiency (FACS results) of mSWCNT-FITCbefore and after 70% ethanol washing. A: 70% ethanol and PBS washing; B:PBS washing only.

FIG. 22 shows Canola and carrot protoplast viability treated withmSWCNT-FITC.

FIG. 23 shows confocal images of canola and carrotprotoplasts/mSWCNT-FITC. (Because the size of carrot cell is muchsmaller than that of canola cell, the green fluorescent signal in carrotcell is weaker than the canola cell.)

FIG. 24 shows sectional TEM images of canola and carrotprotoplasts/mSWCNT-FITC.

FIG. 25 shows the synthesis of iron oxide nanoparticles. (A) Solution ofmagnetic iron oxide nanoparticles. (B) Solution of magnetic iron oxidenanoparticles beside a magnet. We can clearly see the nanoparticles weredriven towards the magnet side. (C) AFM image of 15-20 nm magnetic ironoxide nanoparticles. (D) TEM-scan image of 15-20 nm magnetic iron oxidenanoparticles. (E) AFM analysis showing the vertical height (15 to 20nm) of the nanoparticles in image C.

FIG. 26 shows synthesis of mGNPs: (A) TEM image of mGNPs with a purplecolor. (B) UV-Vis spectrum of mGNPs with a purple color. (C) TEM imageof mGNPs with a red color (D) UV-Vis spectrum of mGNPs with a red color.

FIG. 27 shows core-shell structure of mGNPs. (A) TEM image of mGNPs. (B)Zoomed-in image of an mGNP. (C) EDX analysis of the image in B. (D)Scheme—formation of core-shell mGNP structure.

FIG. 28 shows cytotoxicity of mGNP and FACS results of mGNP-FITCdelivery. (A) KG-1 cell uptake efficiency for mGNP-FITC; (B) Uptakeefficiency comparison of different standing time on Magnet (Purple:control; green: 2 hrs; red: 4 hrs; blue: 6 hrs); (C) Cytotoxicity ofmGNP from MTS method.

FIG. 29 shows images of Fluorescent microscope and confocal for KG-1cell treated with mGNP-FITC. (A) fluorescent microscope (×100), (B)confocal microscope.

FIG. 30 shows synthesis of mGNP-FITC and cell uptake for mGNP-FITC. (A)Synthetic process (B) Cell uptake for mGNP-FITC.

FIG. 31 shows the synthesis procedures of mGNP-FITC

FIG. 32 shows the FITC delivery efficiency (FACS results) andcytotoxicity of mGNPs.

FIG. 33 shows Confocal images of canola and carrotprotoplasts/mGNP-FITC.

FIG. 34 shows sectional TEM images of canola and carrot protoplasts.

FIG. 35 shows sectional TEM images of canola intact cell.

FIG. 36 shows fluorescent microscope images of canola and carrotprotoplasts/mGNP-FITC (×100)

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

This invention comprises a method to deliver biomolecules or othermolecules of interest into cells using a molecular delivery vehicle,which is magnetically drivable and capable of bonding to at least onebio-molecule. This molecular delivery vehicle can pass through the cellwall with the aid of an external magnetic force.

“Biomolecule”—a biological molecule that performs some function whichinfluences the behavior or nature of a biological system.

“Magnetic nanoparticle”—any particle on the nanoscale (having onedimension less than about 100 nm) the motion of which is influenced by amagnetic field.

“Nanoscale”—the range of lengths used to measure objects from 0.1 nm upto 1000 nm where 1 nm is 10⁻⁹ meters.

“Transfect”—a process to introduce foreign genetic material into a cell.

The present invention relates to the use of magnetic nanoparticles totransport biomolecules and other molecules of interest across a cellmembrane.

In one embodiment of the present invention, the magnetic nanoparticlestake the form of a metal core coated in a material such as carbon asshown in FIG. 1B. These nanoparticles are then functionalized so that abio-molecule can be bonded to them.

In one embodiment of the present invention, the magnetic nanoparticlesare carbon nanotubes, such as single-walled carbon nanotubes (SWNT)embedded with magnetic metal atoms (FIG. 1A). In one embodiment, themagnetic metal atoms comprise nickel, iron or cobalt.

Single-walled carbon nanotubes are well known in the art and may besynthesized using any suitable technique, such as chemical vapordeposition technique (CVD). These carbon nanotubes are grown from asurface using nickel or yttrium, or both nickel and yttrium, as seed. Inone embodiment, the nickel and/or yttrium is thus incorporated at leastinto the tip of the carbon nanotube. In one embodiment, suitable SWNTshave a diameter between about 1.2 to about 1.5 nm, and a length of about2 to about 5 μm. The SWNTs may be either armchair or chiral nanotubes.In one embodiment, the SWNTs used are armchair (5,5) nanotubes.

The magnetic nanoparticles or carbon nanotubes are prepared for bondingto a bio-molecule by adding functional groups to them. These functionalgroups act as the bonding site, which will hold the bio-molecule to thenanoparticles or the carbon nanotubes. In addition, functionalization isimportant as many nanoparticles or carbon nanotubes, particularly SWNTs,are insoluble in water. Functionalization increases their watersolubility.

In one embodiment, shown schematically in FIGS. 4A and 4B,functionalization is achieved by chemically altering the surface of thecarbon nanotube. In one example, the surface of the magnetic carbonnanotube is carboxylated and the carboxylic acid is reacted with thionylchloride to provide an acid chloride. The acid chloride may then becoupled with tert-butyl-12-aminododecylcarbamate, or tert-butyl(2-aminoethyl)carbamate, followed by deprotection of the Boc group toprovide the amine derivative.

In an alternative embodiment, amine derivative nanotubes can be producedby reacting the acid chloride nanotube with then2′-(ethylenedioxy)bis(ethylamine) to produce the amine derivative, asshown in FIG. 4C. In a further alternative, the amine derivative may beformed using ethane-1,2diamine, as shown in FIG. 4D.

In one example, the amine derivative is then reacted with fluoresceinisothiocynanate (FITC) giving rise to the FITC derivatized magneticcarbon nanotube.

These magnetic carbon nanotube bonded molecules may then be subjected toa magnetic field and a cell culture, as described herein.

Biomolecules such as DNA or RNA can be attached to carboxyl functionalgroups on the surface of the nanoparticle or carbon nanotube. In oneexample, plasmid vectors may be combined with carboxylated SWNTs and1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) in2-[N-morpholino]ethane sulfonic acid (MES) or a phosphate buffer (pH4.5) for the aminization between the primary amine groups in the DNAmolecules and carboxylic groups on the nanotubes. Alternatively, DNA orRNA can be bound by electrostatic interaction with amine functionalgroups on the surface of the nanoparticle.

The nanoparticles may comprise silica or other materials which may bebiodegradable or biocompatible within a cell, such as, withoutlimitation, nanocellulose, or nanocrystalline cellulose. The term“biodegradable” as used herein means that the substance may be brokendown into innocuous products by the action of living things. The term“biocompatible” means that the substance does not have toxic orinjurious effects on biological function of cells either in vitro or invivo. In one embodiment, a carbon nanotube may be coated with silica andthe carbon then removed or burnt out, leaving a silica nanotube based onthe carbon template. The silica nanotube may then functionalized usingmethods similar to those described herein for carbon nanotube, and asare known to those skilled in the art.

Once the biomolecule or other molecule of interest is bonded to themagnetic nanoparticle, the nanoparticle is placed in a solution alongwith the cells that are to be transfected and a magnetic force isapplied so that the nanoparticles are accelerated through the solution.Inevitably, these will collide with a cell and there will be aprobability that the particle will pass through the membrane into thecell, as shown schematically in FIG. 2. If the particle does not enterthe cell, it will be free to accelerate again to attempt to transfectanother cell. A substantial majority of the cells will be transfectedafter a relatively short period.

The magnetic field that is used to drive the molecular delivery vehiclesis configured so that it provides a magnetic force which can be staticor variable in direction and magnitude. In one embodiment, the magneticfield is configured so that the magnetic force can change between beingvariable and static at different stages of delivery. In one embodiment,the magnetic nanoparticles can be caused to move in complex paths byconstantly varying magnetic force, which is changing its magnitude anddirection.

In another embodiment, the delivery vehicles can be moved in complexpaths and at variable velocities and accelerations.

In one embodiment, the membrane that must be transfected can be mademore permeable by applying ultrasound energy to the cell culture, suchas low-intensity pulsed ultrasound. The ultrasound may be applied athigher frequencies than is known to enhance cell growth. Typically LIPUShas been used at frequencies less than about 1 MHz, however, inembodiments of the present invention, any frequency between 1 MHz to 2MHz may be used, such as 1.5 MHz.

Ultrasound can be applied using conventional or slightly modifiedtherapeutic ultrasound transducers. The intensity of the ultrasoundenergy may vary from 0.1 W/cm² to about 1.0 W/cm². In one embodiment,the intensity is between about 0.3 W/cm² to about 0.5 W/cm². Varyingduty cycles and pulse repetitions may be used, such as a duty cyclebetween about 20% and 100% and a repetition frequency of 100 Hz. Ingeneral, higher intensities and longer duty cycles will increasemovement across cell membranes, at the expense of cell viability. Totalultrasound energy, calculated as follows, should preferably not exceed alevel where cell viability is substantially impaired.

Energy (J)=Intensity*Duty Cycle*Time

In one embodiment, total energy may optimally be 18000 mJ.

Suitable ultrasound contrast agents, such as Definity™ (Bristol-MyersSquibb) may be used to promote microcavitation in the vicinity of thecells.

In one embodiment, the magnetic nanoparticles may be used in vivo todeliver therapeutic agents such as drugs or biomolecules to a specifictarget. A magnet may be placed at the site where the magneticnanoparticles are to be focused, as shown in FIG. 3. As the magneticnanoparticles circulate through the body, they will accumulate at thesite where the magnet is located. Thus, the nanoparticles deliver thebiomolecules to a specific target region.

In one embodiment, this targeted delivery mechanism may be used todeliver molecules into difficult to access areas, such as across theblood-brain barrier into the central nervous system. The magneticnanoparticles can be collected at a specific site of the blood brainbarrier using a magnetic field. Then, using a magnetic force thesenanoparticles can be forced across the barrier.

Once the nanoparticles have been concentrated at a specific point orregion, the nanoparticles can be forced into cells at that site by usinga magnetic force with rapidly alternating direction. This will excitethe particles to move back and forth quickly. As they move around theywill collide with the cell membrane and at least a portion of theparticles will pass through the membrane into the cell. In oneembodiment, the use of ultrasound and magnetic forces may be used toenhance such movement in vivo. Ultrasound transducers which applyultrasound energy to the human body are well known for imaging purposes,and may be used for the molecular delivery systems described herein withlittle or no modification.

The present invention may be embodied in other specific forms withoutdeparting from its structures, methods, or other essentialcharacteristics as broadly described herein and claimed hereafter. Thedescribed embodiments are to be considered in all respects only as is,therefore, indicated by the appended claims, rather than by theforegoing description. All changes that come within the meaning andequivalence of the claims are to be embraced within their scope.

EXAMPLES

The following examples are intended to be illustrative of the describedinvention, and not be limiting of the invention claimed herein, exceptwhere specifically recited.

Example 1 Synthesis of FITC-Labelled Carbon Single-Walled Nanotubes(SWNT) Scheme Shown in FIG. 4B

Nickel containing carbon nanotubes were refluxed with 3N HNO₃ for 45 hto introduce carboxylic acid groups. After refluxing, the solution wasdiluted with deionized water, filtrated and washed several times withdeionized water. The acid treated SWNTs were collected and dried undervacuum.

100 mg of SWNTs were stirred in 20 mL of SOCl₂ (containing 1 mL ofdimethylformamide) at 70° C. for 24 h. After centrifugation, thebrown-colored supernatant was decanted and the remaining solid waswashed with anhydrous tetrahydrofuran. After centrifugation, thepale-colored supernatant was decanted. The remaining solid was driedunder vacuum.

A mixture of the resulting SWNTs and 1 g oftert-butyl-2-aminoethylcarbamate was heated at 100° C. under an argonatmosphere for 100 h. After cooling to room temperature, the excesstert-butyl-2-aminoethylcarbamate was removed by washing with methanol.The resulting black solid was dried under vacuum.

The coupling product of SWNTs with tert-butyl-2-aminoethylcarbamate wassuspended in MeOH and a solution of HCl in dioxane (4 N) added, theresulting mixture was stirred at room temperature for 4 h. Thenanhydrous ethyl ether was added, the resulting precipitate was collectedand dried under vacuum.

The amine groups-containing SWNTs were suspended in a mixture of DMF anddiisopropylethylamine and a solution of fluoroisothiocyanate (FITC) inDMF was added. The resulting mixture was stirred for 4 h at roomtemperature in darkness. Then anhydrous ethyl ether was added, theresulted precipitate was collected by centrifugation and washedthoroughly with ethyl ether and methanol, dried under vacuum to giveFITC-labeled SWNTs.

In an alternative method, shown schematically in FIG. 4C, SWNTs fromAldrich were oxidized to form carboxylic acid groups on the surface.These nanotubes were reacted with thionyl chloride and then2′-(ethylenedioxy)bis(ethylamine) to produce amine-terminated nanotubes.The amine was then reacted with FITC to attach FITC to SWNTs.

Example 2 IR, XPS and UV-Vis Characterization

To validate the all synthesis take place, all of the intermediates shownin FIG. 4C and final product (SWNT-FITC) were characterized by Infrared(IR), X-ray photoelectron spectroscopy (XPS) and UV-vis and the resultsare shown in FIGS. 5 and 6. IR data clearly show that SWNTs weresuccessfully functionalized to give carboxylic groups and XPS data showthat about 6.1% of the carbon atoms are present as carboxyl groups. TheUV-vis spectrum of the FITC-labeled SWNT in water is shown in FIG. 8,for comparison, the UV-vis spectrum of the FITC in water is shown in thesame figure.

Example 3

Fluorescent Staining and Imaging FITC-Labeled SWNTs (CNT-FITC) asPrepared using the method described in Example 1 (FIG. 4B) were used tostain and image a human breast adenocarcinoma cell.

Materials

-   -   Cell—MCF-7    -   Medium—GIBCO 11330, DMEM/F12 (1:1)    -   Formaldehyde Solution (w/v) 16%, Methanol-free, Pierce,        Cat#28906    -   Hoechst—Invitrogen Cat#33342    -   Rhodamine Phalloidin—Invitrogen Cat# R-415

(Rhodamine Phalloidin 300U was dissolved in 1.5 ml Methanol to formconcentration of 200 units/ml, distributed them into 10 μl each vial,store at −20° C.)

-   -   PBS buffer    -   Block buffer—PBS/0.5% BSA    -   Magnets—Applied Magnets Cat#ND075 (www magnet4less.com) 2×1 in        thick disc, Grade N42, Rare earth Neodymium super strong magnet        (Pull force: 176 lbs)

Round cover slips were placed into a 6-well or 24-well plate, one coverslip into one well and MCF-7 cells into each well, cell number:1×10⁵/ml, and incubated at 37° C. over night. Add Hoechst into each well(1 μl Hoechst in 1 ml medium) and incubate at 37° C. for 1 h. 1 ml ofCNT-FITC was added into each well of the plate (except the control) andincubate at 37° C. for 1 h. Each well was washed 3 times with PBS.

A cover slip picked out of one well with tweezers, and verticallyinserted into a beaker containing 10 ml serum-free medium supplementedwith CNT-FITC (10:1, medium: CNT-FITC) was placed on hotplate (magneticstirrer) with the cells facing the incoming nanotubes for 3 min. Thespeed of the stirrer was set at 1,200 rpm. The cover slip was laid onone dish containing serum-free medium without CNT-FITC, and the dish wasplaced on a magnet for 7 min. The cover slip was then washed 3 timeswith PBS and placed in another 24 well plate, along with cover slipswhich were not placed on a magnet.

The cells were fixed with 4% Formaldehyde Solution for 10 min (or overnight at 4° C.). The formaldehyde solution was removed and the cellswashed 3 times with PBS. 250 μl of PBS/0.2 TX-100 was added onto thecover slips in the wells and place at room temperature for 10 min. Againthe cells were washed 3 times with PBS, and blocked with 250 μl ofPBS/0.5% BSA for 20 min. 2.5 μl Rhodamine Phalloidin was added to 50 μlblock buffer and the mix pipetted on parafilm. The cover slip wasoverlaid onto the solution in place for 30 min.

The cover slips were then placed back to the plate and washed 3 timeswith PBS. The coverslips were then mounted onto slides and send for theconfocal microscopy. Samples were imaged with a laser scanning confocalmicroscopy 510 (Carl Zeiss) equipped with Axiovert 100M microscopy(Zeiss), a F-Fluar 40×-1.3 NA oil lens and 3 different lasers (Uv,Argon/2 and HeNe1).

As shown in FIGS. 7A and 7B, the cell nuclei fluoresce strongly as aresult of the Invitrogen stain which combines with double-stranded DNA.In FIG. 8B, fluorescence of the FITC moities may be plainly seen withinthe cells cytoplasm, indicating that the CNT-FITCs have passed throughthe cell membranes and into the cytoplasm.

In another example, SWNT were conjugated to GFP plasmid (pDRIVE5-GFP) bycovalent bonding using EDC and a phosphate buffer. The SWNT-GFP plasmidwas then incubated with MCF-7 cells for 3 min, followed by 7 minuteswith a magnetic field supplied by a magnetic stirrer. The cells werethen incubated for 24 hours and confocal microscopy was used to confirmGFP expression. FIG. 7D shows results of GFP fluorescence within thecells, as compared to the control cells in FIG. 7C.

Example 4 Cell Uptake Efficiency

FITC-labeled SWNT was delivered into adherent MCF-7 breast cancer cells.Following the delivery and recovery phases, the fluorescently-labelledSWNT was detected by confocal microscopy. The results are presented inFIGS. 8A and 8B. The data clearly shows that the SWNT crossed the cellmembrane and entered the cell cytoplasm and even into the nucleus (referto the green dots in FIG. 8B; some of them are pointed by the arrows).The uptake rate is about 90% shown in FIG. 9.

In addition to delivery of FITC to adherent cells, like MCF-7 cells, wealso successfully delivered FITC into difficult-to-transfect cells, orsuspension cells, like hematopoietic stem cells (HSCs). FIG. 10 showsthe delivery results. The results show that SWNT can deliver FITC intoHSCs. As time increases to 3 and 6 hours, more FITC enters the cell(FITC shows as green fluorescence). The control sample showed nointernal fluorescence.

Example 5 Cell Viability

Furthermore, it is worth noting that cell viability was not compromisedby SWNT uptake when compared with control, as shown in FIG. 11.Viability of MCF-7 cells after FITC-SWNT uptake with exposure to amagnetic field was compared to the control cells and cells exposed toSWNT alone with no magnetic field. Cells exposed to SWNT appear tosubstantially similar to control populations for viability after 6hours.

Example 6 Ultrasound Delivery (USD) Cell Preparation and DNA

USD and transfection was assessed using human breast adenocarcinomacells (MCF-7). Cells were maintained in the IMDM medium with 10% fetalbovine serum. Cells were harvested a day before the experiment by adding0.25% Trypsin to the culturing flask and waiting for detachment. 1 mL ofcells was added to 10 mm×35 mm dishes with an additional 1 mL of medium.Cell concentration was approximately 1.5×10⁵ cells/mL. To determinetransfection, green fluorescence protein plasmid (GFPplasmid-pDRIVE5-GFP) was added to the medium 15 minutes beforesonication. Various concentrations of GFP were used: 2 μg/mL, 15 μg/mL,and 30 μg/mL. The ultrasound contrast agent Definity, purchased fromLantheus Medical, was used to promote cavitation. The UCA volume usedwas 140 μL.

USD was performed using the Excel UltraMax therapeutic ultrasoundmachine, probe radius 2.5 cm. The ultrasound probe was coupled to thebottom of the cell dish using ultrasound gel. Ultrasound was applied for60 seconds, at a 1 MHz frequency with varying output intensity: 0.3W/cm², and 0.5 W/cm². The duty cycle was tested at 100% or 20% with afixed pulse-repetition frequency of 100 Hz. As controls, we sonicatedblank samples with no UCAs or GFP, and samples with GFP but no UCAs.Additionally, we ran a positive control using PEI, a lipofection agent.Finally, we prepared a sample that was not stimulated by ultrasound, butcontained both Definity and GFP.

Cell counting was conducted in a fluorescence-activated cell-sorting(FACS) machine. 24 hours after USD, cells were collected in FACS testtube with 0.25% trypsin and washed once with 1×PBS. After all above,cells were resuspended in 200 uL 1% paraformaldehyde and tested throughflow cytometry.

Cell viability was assessed by a cell count using a hemacytometer. Aftercollecting cells in the FACS test tube, transfer 20 μl of each sampleinto small centrifuge tubes and dilute with 0.4% trypan blue. Put 10 μlin the hemacytometer and count cell number. Finally calculate the cellconcentration with the following formula: Cell number counted in allsquares/total number of squares counted*dilution factor*1×10⁴.

All the FACs test results are shown in FIGS. 12 to 15. Our negativecontrol samples did not yield any transfection, but maintained excellentcell viability, as seen in the FACs result. The PEI lipofection positivecontrol showed GFP expression, and extreme cell death.

TABLE 1 Transfection results of positive and negative control GFPDefinity W/cm²-DC- % [μg] [μL] sec Transfection 1. 0 0 0-0-0 0.16% 2. 20 0-0-0 0.29% 3. 2 + PEI 0 0-0-0 33.12%

FACs analysis shows that as the exposure intensity increased the cellviability decreased. The maximum transfection was seen with an outputintensity of 0.5 W/cm² and a 20% duty cycle, at 32.51%. Cell viabilityis significantly lower at the output intensities above this level. Thisresult suggests that the output energy achieved by a 0.5 W/cm² and a 20%duty cycle, for 60 seconds is optimum for effective transfection.

The effect of DNA concentration on transfection efficiency was examinedat every energy level. In every case, increasing the DNA concentrationleads to an increase in transfection.

TABLE 2 Transfection results for varied ultrasound output intensity, andGFP concentration. GFP Definity Output intensity, [μg] [μL] Duty cycleTransfection % 2 140 0.5 W/cm², 20%  16.20% 15 140 0.5 W/cm², 20% 26.93% 30 140 0.5 W/cm², 20%  32.51% 2 140 0.3 W/cm², 100% 7.52% 15 1400.3 W/cm², 100% 9.71% 30 140 0.3 W/cm², 100% 14.67% 2 140 0.5 W/cm²,100% 19.63% 15 140 0.5 W/cm², 100% 26.76% 30 140 0.5 W/cm², 100% 32.12%

MCF-7 cells were used to evaluate the effects of ultrasound on genedelivery. We found that the efficiency of ultrasound mediated genedelivery, depended on plasmid concentration, while the viability of thecells was directly related to the ultrasound's output intensity. Thelatter could be due to the fact that the other physical effects ofultrasound, such as transient increase of local temperatures andpressure, are detrimental to cells, or that the pores the cavitationeffect opened were unable to re-seal.

The results from the negative control samples show that the DNA plasmidGFP is unable to diffuse across the cell membrane on its own. The USDresults show that the application of ultrasound with UCAs allow the DNAplasmid to transfect and be expressed by the cell. Furthermore, ourresults demonstrate that there is an optimum ultrasound exposure levelfor transfection and cell viability; the existence of optimum exposureparameters is consisted with other literary results. The FACs resultsexhibit that any output energy greater than 18000 mJ is detrimental tocell viability, where:

Energy (J)=Intensity*Duty Cycle*Time

Due to the nature of the FACs analysis, the transfection resultsobtained from the 0.5 W/cm², 100% duty cycle sample may be skewed. Sincea high percentage of cells in this sample were dead, transfectionpercentage we obtained is misrepresented and cannot be compared to ourresults obtained with higher cell viability.

Plasmid concentration was an important factor in determiningtransfection efficiency. In every case, transfection rate increased withDNA concentration. This result leads us to consider the importance ofDNA proximity to the cells during USD. However, it is expected that theeffect of increasing plasmid concentration to increase transfectionefficiency will eventually plateau.

The findings from the lipofection agent, PEI, revealed two results.First, it confirms that the plasmid GFP can be expressed by the MCF-7cells, but more importantly it highlights the importance of USD. TheFACs results show an extremely high amount of cell death due to PEI. Incontrast, USD was able to obtain similar transfection efficiency whilemaintaining a much lower cell death rate.

Example 7 Formation of Silica Nanotubes

An amount of magnetic single-walled carbon nanotube powder was mixedwith ground Na₂SiO₃.9H₂O (Na₂SiO₃.9H₂O/carbon nanotube ratio was 0.2 byvolume). The mixture was ground carefully for 10 min to mix thereactants uniformly. Excessively ground NH₄Cl (NH₄Cl/Na₂SiO₃.9H₂O=3 byvolume) was then added to the mixture. After being ground carefully for50 min, the product was aged for 5 h and then washed three times withdistilled water. Silicon dioxide coated nanotubes (Si-NT) were obtainedafter being dried at 60° C. for 5 h.

Particles core level spectra were measured using X-ray photoelectronspectrometer (VG ESCALAB MK II). The excitation source was a Mg X-rayanode and HV equalled to To determine crystallite sizes and phase purityof the powders, the X-ray diffraction spectrum was obtained with aRigaku D/max-rA X-ray diffractometer using Cu Kα (λ=1.54056 A)radiation.

Si-NT′ morphology was observed with JEOL JEM 2010 transmission electronmicroscope (TEM) operating at 200 kV, as shown in FIG. 16. TEM sampleswere prepared by dispersing a small amount of powder in ethanol. A dropof the dispersion was then transferred onto coated grid and died forobservation.

Example 8 Si-NT Functionalization

Oxidation of the Si-NTs: The Si-NTs (200 mg) were refluxed to introducecarboxylic groups. After refluxing, the solution was diluted withdeionized water, filtered over a 0.2 μm polycarbonate filter (Millipore)and washed several times with deionized water. The sample was collectedand dried overnight in a vacuum oven at 800C to give Si-NT-2 (170 mg).

The carboxylated Si-NT underwent IR spectrum analysis, with the resultsshown in FIG. 17.

Reaction with thionyl chloride to give Si-NT-COCl: A suspension ofSi-NT-2 (100 mg) in 20 mL of SOCl₂ together with 5 drops ofdimethylformamide (DMF), was stirred at 70° C. for 24 h. The mixture wascooled and centrifuged at 2000 rpm for 30 min. The excess SOCl₂ wasdecanted and the resulting black solid was washed with anhydrous THF(3×20 mL), dried overnight in a vacuum oven at 80° C. to give Si-NT-3(78 mg).

Coupling with ethylenediamine: The mixture of Si-NT-3 (50 mg) andanhydrous ethylenediamine (120 mL) was heated at 100° C. for 100 h.During this time, the liquid phase became dark. After cooling, themixture was poured into methanol (100 mL), centrifuged to give a blacksolid, which was washed several times with methanol. The resulting solidwas dried overnight in a vacuum oven at 80° C. to give Si-NT-4 (42 mg).

Functionalization with GFP plasmid: A suspension of the Si-NT-4 (25 mg)and GFP plasmid (5 mg) in anhydrous DMF (10 mL) was stirred in dark for5 h, then the reaction mixture was poured into anhydrous ethyl ether (40mL), centrifuged to give a black solid, which was washed with methanoluntil TLC (10% MeOH in dichloromethane) showed no free GFP left. Theproduct was dried overnight in a vacuum oven at 80° C. to get the finalproduct (23 mg), Si-NT-GFP.

Example 9 Transfection of HeLa

HeLa cells were grown in RPMI 1640 supplemented with 10% FB in 35 mmPetri dish with a cover slip.

Si-NT-GFP solution was prepared by weighing 3 mg Si-NT-GFP powder into50 ml centrifuge tube. 3 ml of sterilized DI water was added andsonicated until the silica tube powder dissolve and incubated for 1 hrat room temperature. The final volume was brought to 50 ml using RPMI1640 medium w/o FBS. A similar solution with Si-NT was prepared as acontrol. The test and control silica tube solutions were added to 100 mlbeakers.

200,000 cells were seeded per dish and cultured overnight allowing cellsto attach. A volume of test or controls solutions were added to thedishes and the cells were then magnetically treated for 3 min verticallyby putting dishes on top of magnetic stir hot plate and followed by 7mins with Petri dishes on top of a stirring magnet.

The cells were washed twice with PBS, and replaced with 2 ml of culturemedium. The dishes were returned to incubator and incubated for 24 hrand 48 hr, respectively.

Each of the samples were prepared for and viewed with confocalmicroscope observation of the GFP signal. The results are shown in FIG.18.

Toxicity studies showed that increasing concentrations of Si-NT hadlittle effect on cell survival rate, as shown in FIG. 19.

Example 10 FITC Delivery into Plant Cells Using Magnetic Single-WallCarbon Nanotubes

Experiments and Methods

Cell culture: MD cell suspensions of canola (B. napus L. var. Jet Neuf)are maintained on a rotary shaker (160 rpm) at 20° C. in NLN media (pH6.0, containing 6.5% sucrose, 30 mg l⁻¹ glutathione, 800 mg l⁻¹glutamine, 100 mg l⁻¹ L-serine, 0.5 mg l⁻¹ a-naphthaleneacetic acid(NAA), 0.05 mg l⁻¹ ⁶-benzylaminopurine (BA) and 0.5 mg l⁻¹ 2,4-D) (13).At 2-week intervals, one third of the mass of cells grown in 125 mlflasks is transferred to 50 ml of fresh NLN medium. Seeds of carrot (D.carota L. var. Konservnaja 63) are obtained from Plant Gene Resources ofCanada (Saskatoon, Saskatchewan). Cells derived from leaves of in vitroplants are cultured in MS media, 3% sucrose, 0.2 mg l⁻¹ BA, 1.0 mg l⁻¹NAA (pH=6). Two to Three days after subculture, cells are used forprotoplast isolation.

Protoplast isolation: Plant cells are preplasmolyzed by incubation inCPW13M solution for 1 h at room temperature. The solution was thenreplaced with a digestion solution, consisting of ½ MS salts, 0.06%2-(N-Morpholino)ethanesulfonic acid (MES), 13% mannitol, 0.1% MacerozymeR-10 (Yakult Honsha Co., Japan) and 0.5% Cellulase Onozuka R-10 (YakultHonsha Co., Japan), pH 5.8. The incubation is carried out overnight (16h) at 25° C. in the dark. The digestion mixture was filtered through asterile nylon cell strainer (40 μm, BD Falcon, USA) to remove thedebris, and then centrifuged (100×g) for 10 min. The pellet wasresuspended in CPW255 and 2 ml of CPW13M was added to the top. Theprotoplasts are then collected with sterilized Pasteur pipettesfollowing centrifugation (100×g) for 10 min, washed twice, and finallyresuspended in ½ NLN medium supplemented with 13% mannitol. Theprotoplast solution was used for the mSWCNT-FITC delivery experiment.

Synthesis of mSWCNT-FITC: 2 mg of purified mSWCNTs is dissolved into a120 ml flask containing 5 ml of concentrated H₂SO₄/HNO₃ (V:V=3:1). Thesolution is sonicated for 10 minutes, and then washed completely. ThemSWCNTs are resuspensed into a 120 ml flask containing 200 ml of MilliQwater. 5 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbondiamidehydrochloride (EDC) and 1 ml of ethyl diamine are added into the flask.The mixture is stirred for 30 minutes in the dark. The solution isdialysed until no free ethyl diamine and EDC remained in solution. 100mg of FITC is dissolved into 10 ml of DMF and added into the dialysedsolution. The mixture is stirred for 5 minutes and kept at roomtemperature overnight. The mixture solution is dialysed until no freeFITC molecules remained in solution.

Magnetic-field-driven cellular uptake experiment: Protoplasts with adensity of 5×10⁵ cells/plate are placed in 35 mm culture dishes and thedishes are sealed with parafilm. The magnetic-field-driven deliverymethod is carried out by placing the culture dishes containing 1 ml ofmedium with 0.25 μg/ml mSWCNT-FITC or mSWCNT on the top of an Nd—Fe—Bpermanent magnet for 12 h, then the protoplasts are collected, fixed in2% paraformaldehyde and completely washed twice with PBS and 70%ethanol.

Cell viability: Protoplasts are seeded in 35 mm Petri dishes in culturemedium. 30 μl of mSWCNTs is added into each dish. The Petri dishes areput on top of the Nd—Fe—B magnet at room temperature overnight. A dropof cell solution is deposited on a glass microscope slide and stainedwith FDA. Images are taken with both bright and green channels under afluorescent microscope (Leica CW 225 A with Nikon digital cameraDXM1200). The number of protoplasts is counted under bright channel andfluorescent channel. Then cell viability or NPs cytotoxicity iscalculated.

Flow Cytometry Measurement: Protoplasts exposed to mSWCNT-FITC atdifferent concentrations are collected and centrifuged at 1000 rpm for10 min. The collected cells are extensively washed using PBS, and thenfixed in 2% paraformaldehyde. The fixed cells are washed with 70%ethanol twice again, and then resuspended in 400 μl PBS. The mSWCNT-FITCdelivery efficiency is evaluated with Flow Cytometry (FACscan,Becton-Dickinson, San Jose, Calif., USA) at an excitation wavelength of488 nm.

Atomic force microscope (AFM) imaging: A small amount of sample solutionis directly transferred dropwise onto a silicon wafer. The sample iscovered and kept at room temperature until the solution is dry. AFMimages are taken using a Veeco Multimode V SPM operating in tappingmode.

Confocal microscopy imaging of plant cells: Protoplasts are seeded at adensity of 1×10⁵ cells/cm² on cover slips previously coated withpoly-L-lysine (10 μg/ml) for 45 min. The protoplasts are exposed to 0.25μg/ml mSWCNT-FITC and mSWCNT alone (the control) on an Nd—Fe—B permanentmagnet. After 12 hours of incubation on an Nd—Fe—B permanent magnet, thecells are fixed in 2% paraformaldehyde and washed twice with PBS bufferand twice with 70% ethanol. The sample is examined under a confocallaser scanning microscope (Quorum Wave FX-Sinning Disk) equipped withimaging software—Hamamatsu EMCCD (C9100-13).

TEM imaging: TEM images are taken using a Philips-FEI Morgagni 268instrument operated at 80 kV. The sample solution is deposited on acopper support, which is coated with carbon. Protoplasts are fixed in 2%glutaraldehyde in 4% PEA/cacodylate buffer, pH 7.2, for 2 hours at roomtemperature. (a) The fixative solution is drained off and replaced with0.1M PBS buffer. Two further changes are done 10 minutes apart. (b) Thebuffer is drained off and the sample is post-fixed with 1% osmiumtetroxide (OSO₄ in 0.12 M Cacodylate buffer, pH 7.2) for one hour. (c)The sample is washed using 0.1M phosphate buffer 3 times for a total ofone half hour. (d) The sample is dehydrated through a graded ethanolseries as follows: 50%, 70%, 90%, 100×3 changes; one change every 15minutes. (e) The ethanol is drained off from the specimen and newethanol: Spurr mix is added for 3 hours. The ethanol: Spurr mix isreplaced with pure Spurr resin. The Petri dish is sealed overnight. (f)The Spurr resin is replaced again and the sample is dried at 70-80° C.in an oven for 18 hours. (g) The sample is cooled and then removed frommolds. (h) The sample is ultracut by a Reichert-Jung Ultramicrotome andstained with uranyl acetate and lead citrate.

Synthesis of mSWCNT-FITC

Nickel nanoparticles remained on the surface or are trapped insideSWCNTs after purification (black dots in FIG. 20C), which indicates thatthese nanotubes are magnetic SWCNTs (mSWCNTs). After purification, thesemSWCNTs still exist in bundles with diameters ranging from about 20-40nm (FIGS. 20A, 20B), which suggests at least 10 SWCNTs are bundledtogether because the diameter of a single SWCNT is about 2-3 nm. FIG.20D shows the synthetic process for making mSWCNT-FITC. The mSWCNTs arelinked with FITC covalently through the linkage of ethyl diamine, andthis covalent bond ensured that during the mSWCNT delivery process, FITCmolecules and mSWCNTs are not separated.

According to FIG. 21, based on the FACS analysis results, FITC deliveryefficiency is about 100% for both canola and carrot protoplasts whenmSWCNT concentration is in the range of 0.06-0.25 μg/ml. For both canolaand carrot protoplasts, a higher concentration of mSWCNTs results in astronger fluorescence signal. This result shows that higher mSWCNTconcentration corresponds to more mSWCNT-FITC entering cells. In orderto ensure the mSWCNT-FITC which is attached to the surface of cells iscompletely removed, the protoplasts are washed twice using 70% ethanolafter washing twice with PBS. In FIG. 21B, the protoplasts are washedtwice only using PBS. Compared with the FACS results in FIG. 21B, afterwashing with ethanol, there is a small left shift for canola protoplastsand a larger left shift for carrot protoplasts are observed (FIG. 21A),which indicates most mSWCNT-FITC outside of the cells are washed awaybecause SWCNTs are more soluble in ethanol than in water. Thedistributive curves of cell counts from the controls in FACS aredifferent from that of normal mammalian cell lines. For instance, thefluorescent strength of normal mammalian cell lines is on the order of10⁰-10¹, but those of the two protoplasts tested are on the order of10⁰-10², which indicated some of the fluorescent signal is from theprotoplasts.

It is hypothesized that these results mainly come from the remainingcell walls that the enzyme used could not completely remove. In fact,this assumption is confirmed by fluorescent microscope because somefluorescent signals from the cell walls of canola cells can be observed.Although the fluorescent signal from the cell walls interferes with theFACS results, it is still seen that all fluorescent signals becomesstronger after mSWCNT-FITC delivery. It seems that mSWCNT-FITCpenetrates the cells with or without the cell wall because if nomSWCNT-FITC would have entered into the walled cells, the fluorescentsignals of these walled cells would remain un-shifted.

FIG. 22 shows that mSWCNTs are not cytotoxic for the canola and carrotprotoplasts because the cell viability after treatment with mSWCNT-FITCremained similar to the control.

In order to confirm our observations, confocal and sectional TEM imagingof these two protoplasts is performed. Compared to the control cells,green fluorescent signals appears in most cells after mSWCNT-FITCdelivery. The signal strength is different for different cells, whichreflects how much FITC enters the cells (FIG. 23). Even though thecarrot protoplasts are smaller than the canola protoplasts, themSWCNT-FITC is also able to enter them. There are some green fluorescentsignals which appears near the nucleus, which means that the FITC isnear the nucleus. FIG. 24 is the sectional TEM images of these twoprotoplasts. For canola protoplasts, the mSWCNTs are found in endosomes(FIG. 24-canola A, B, C, D). However, for carrot protoplasts, an mSWCNTis found outside the cell and an mSWCNT is found near nuclear membrane.All these results show that mSWCNTs not only enters cells but alsodistributes in different organelles inside plant cells.

To ensure the delivery of FITC, it is covalently bound with mSWCNTs.FACS results show that mSWCNT-FITC can enter canola and carrotprotoplasts driven by an external magnetic force. The FITC deliveryefficiency is about 100% according to FACS results. Confocal andsectional TEM images further confirm that mSWCNT-FITCs are inside theseplant cells. mSWCNTs are also found both in the endosomes of canolaprotoplasts and outside endosomes near the nuclear membrane of carrotprotoplasts.

Example 11 Magnetic Gold Nanoparticles Synthesis, Characterization andits Application in the Delivery of FITC into KG-1 Cells

Materials and Methods

Chemicals: The sodium citrate trihydrate, chloroauric acid, ascorbicacid, fluorescein isothiocyanate (FITC), dimethylformamide (DMF) andsodium dodecyl sulfate (SDS) used in this study are from Sigma-Aldrich.Iscove's Modified Dulbecco's Medium (IMDM), Fetal Bovine Serum andPenicillin/streptomycin used are from GIBCO. Thiol polyethylene glycol(PEG) with amino functional group is purchased from NANOCS company withmolecular weight 5000.

Cells: KG-1, acute human leukemia cell lines are purchased from theAmerican Type Culture Collection (ATCC HTB22, Rockville, Md. USA).

Synthesis of magnetic gold nanoparticles (mGNPs): The followingprocedures outline the synthesis of mGNPs. (1) Synthesis of ironnanoparticles: 2.78 g of Iron(II) sulfate heptahydrate and 3.25 g ofIron(III) chloride hexahydrate are transferred into to a clean 125 mLconical flask containing 25 mL of MilliQ high purity de-ionized water.0.85 mL of concentrated HCl is transferred into the flask. This solutionis added dropwise into 250 mL of 1.0 N NaOH solution until a blacksolution is obtained. 400 μL of the black solution is diluted to 80 mLusing MilliQ high purity de-ionized water, and is sonicated for 2 hours.(2) Synthesis of mGNPs: 1 mL of 25 mM chloroauric acid and 2 mL of 20%sodium dodecyl sulfate solution (SDS) are transferred to a clean 20 mLvial containing 16 mL of MilliQ high purity de-ionized water. 1 mL ofiron nanoparticle solution prepared above and 300 μL of the above HAuCl₄solution are transferred into a 20 mL vial. The vial is sonicated for 15min. Meanwhile, a solution of ascorbic acid (AA) is prepared bydissolving 0.0400 g of AA powder in 20 mL of MilliQ water. 180 μL of AAsolution is transferred into the vial and stirred for 30 min. 200 μL of10% HCl solution is transferred into this vial and stirred for anadditional 30 min.

Synthesis of mGNP-FITC: (1) 0.0116 g HS-PEG-NH₂ (MW 5000) is dissolvedinto a 20 mL vial containing 10 mL of MilliQ water. 1 mL of the abovemGNP solution is transferred into this vial and stirred for 5 min. Thisvial is kept at 4° C. overnight. (2) The solution is centrifuged at10000 rpm for 30 min. The supernatant is discarded and the sediment iswashed once using the same centrifuge conditions. The sediment isdissolved in 0.5 mL of MilliQ water (mGNP solution). Meanwhile, 100 mgFITC is dissolved into 0.5 mL of DMF, and then mixed with above mGNPsolution. The mixture is stirred for 5 minutes before being kept at roomtemperature overnight. The mixture is dialyzed until no free FITC insolution remained.

Cell Culture and Magnetic-Field-Driven Cellular Uptake Experiment

KG-1 cells with a density of 5×10⁵ cells per plate are placed inpoly-L-lysine (10 μg mL⁻¹)-coated 35 mm culture dishes and incubated for45 min at 37° C., 5% CO₂. The magnetic-field-driven delivery method isto place a culture dish containing 1 mL IMDM media with 18.8 nmol AumL⁻¹ of mGNP-FITC or mGNP on the top of an Nd—Fe—B permanent magnet for2-6 hrs, then the culture dish is put back in incubator overnight. Theuptake experiment is terminated by washing the cells with PBS buffer.

MTS experiment: (1) 30,000 cells are seeded per well in 96-well plates.The experiment is conducted in quadruplicate. (2) mGNP stock solution isdiluted in growth medium to concentrations of 4.7, 9.4, 18.8, 37.5, and75 nmol Au mL⁻¹. (3) 200 μL of mGNP-FITC containing growth medium isadded per well and the 96-well plates are put back into the incubator tocontinue culture for 24 and 48 hrs. (4) 20 μL of MTS solution is added(5 mg mL¹ in 1×DPBS), then the cells are incubated for additional 3 hrs.(5) Absorbance at 490 nm is measured.

Flow Cytometry Measurement: KG-1 cells exposed to mGNP-FITC fordifferent amounts of time on magnets are collected and centrifuged at1200 rpm for 10 min. The collected cells are extensively washed usingPBS and then fixed in 1% paraformaldehyde and resuspended in 400 μL ofPBS. The mGNP-FITC delivery efficiency is evaluated with Flow Cytometry(FACscan, Becton-Dickinson, San Jose, Calif., USA) at an excitationwavelength of 488 nm.

Atomic force microscope (AFM) image: A small amount of sample solutionis directly transferred dropwise onto a silicon wafer. The sample iscovered and kept at room temperature until the solution is dry. AFMimages are taken using Veeco Multimode V SPM operating in tapping mode.

Fluorescent microscopy: The fluorescent images are taken by usingFluorescent Microscopy of Leica CW 225 A with Nikon digital cameraDXM1200.

Confocal microscope images: KG-1 cells are seeded at a density of 1×10⁵cells cm⁻² on cover slips previously coated with poly-L-lysine (10 μgmL⁻¹) for 45 min at 37° C., 5% CO². The cells are exposed to 18.8 nmolAu mL⁻¹ mGNP-FITC and mGNP (the control) on an Nd—Fe—B permanent magnet.Uptake is terminated by washing the cells twice with ice-cold PBS. After4 hrs of incubation on an Nd—Fe—B permanent magnet, the cells isincubated in an incubator for an additional 12 hours, then fixed in 2%paraformaldehyde, stained and examined under a confocal laser scanningmicroscope (Quorum Wave FX-Sinning Disk) equipped with imagingsoftware—Hamamatsu EMCCD (C9100-13).

TEM image: The TEM images are taken using Philips-FEI Morgagni 268instrument, and operated at 80 kV. The sample solution is deposited onthe copper support coating with carbon.

Synthesis of mGNPs

The synthesis of mGNPs consists of two steps. The first step is tosynthesize iron oxide nanoparticles with suitable size. FIG. 25 showsthe design process and characterization of the iron oxide nanoparticles,and we followed this typical method to synthesize iron oxidenanoparticles. The color of the iron oxide nanoparticle solution isblack (FIG. 25A). When a magnet is put beside the solution, the ironoxide nanoparticles quickly move towards the magnet (FIG. 25B), whichconfirms the magnetism of the iron oxide nanoparticles. The iron oxidenanoparticles are big enough that their migrated towards magnet isvisually observable. However, their size is too big for the creation ofmGNPs, and smaller particles have to be prepared. This problem can besolved by using sonication. After sonication treatment, theblack-colored solution of magnetic iron oxide nanoparticles becomeslight yellow (in the middle of FIG. 25). According to AFM and TEM images(FIGS. 25C and 25D), the size of iron oxide nanoparticles is about 15-20nm. The morphology is uneven. AFM analysis of the vertical height of theparticles also gave a similar result (FIG. 25E). The second step is tosynthesize mGNPs. The synthesis of these mGNPs is shown in FIG. 26. Bysonicating the mixture of HAuCl₄, surfactant SDS (sodium dodecylsulfate) and the 15-20 nm iron oxide nanoparticles, the gold cations areadsorbed on the surface or trapped inside the micropores of the ironoxide nanoparticles. After quick reduction, the gold cations becomesgold nanoparticles and aggregated together due to the instability ofnanoparticles. Some aggregated gold nanoparticles form a shell outsidethe iron oxide nanoparticles; some aggregated together surrounding ironoxide nanoparticles (FIG. 25A). The solution is purple and theabsorbance in the UV-Vis spectrum is at 556 nm, which indicates thenanoparticles are bigger (FIG. 25B). The size indicated using thisUV-Vis spectrum method should reflect the mean size of the gold, ironoxide and their aggregation. After the purple solution is treated usinga 5% HCl solution, the solution became red and the absorbance in UV-Visspectrum is at 532 nm, which indicates that the nanoparticles are about20-30 nm in size (FIG. 25D) according to the normal UV spectrumcharacter of gold nanoparticles. During this treatment, the iron oxideoutside the nanoparticles are dissolved and removed; only the iron oxideinside the nanoparticles remains. Therefore, the cluster of iron oxideand gold nanoparticles is broken, the aggregation of nanoparticlesbecomes dispersed into smaller nanoparticles. Because the metallic goldis formed on the surface or inside the micropores of iron oxidenanoparticles, the only iron oxide remaining must have been inside goldnanoparticles. The morphology and size of mGNPs became consistent (FIG.26C). The ideal configuration would be for the metallic gold aggregatedto form a shell around the surface of iron oxide nanoparticles. Thisstructure is confirmed by the zoomed-in image (FIG. 27A, B). Thecore-shell structure of the mGNP can be clearly seen. There is arelative black shell and relative gray core. Because the contrasts ofgold and iron are different in TEM image and the contrast of gold islarger, the black shell in this zoomed-in image should belong to goldand relative gray core should belong to iron oxide. Due to the sphericalstructure, there is a small amount of darker coloring in relative graycore produced by outside gold. The EDX analysis in FIG. 27C shows thatthe nanoparticles are composed of Fe and Au, which verified thecore-shell structure. The schematic of the core-shell structure formingprocess for the mGNPs is shown in FIG. 27D.

FITC Delivery into KG-1 Cell Line Using mGNPs

FIG. 30A shows the linking process between mGNP and FITC molecules; andFIG. 30B shows the cellular uptake experiment design of delivering FITCinto KG-1 cells driven by an external magnetic force. By takingadvantage of the gold covering the magnetic nanoparticles, PEG can becovalently bound with mGNPs because thiol-PEG with amino functionalgroups can interact with gold through thiol functional groups. An FITCmolecule can react with an amino functional group to form a covalentbond through an amide (FIG. 30A). Therefore, through PEG bridges, FITCmolecules can link to the surface of mGNPs through covalent bonds whichcan avoid the FITC lost during uptake process. Due to the solubility ofPEG, mGNP-FITC can dissolve in the culture medium of the KG-1 cell lineto form a uniform solution. Therefore, after the cellular uptakeexperiment, most of the mGNP-FITC left on cell surfaces can be removedby completely washing the cells twice using PBS buffer, then thefluorescent signals in FACS measurement should only come from themGNP-FITC inside KG-1 cells. When the KG-1 cells with mGNP-FITC inculture medium are put on top of the magnet, the mGNP-FITC moves towardsthe bottom of culture dish and is adsorbed on the surface of KG-1 cells.These mGNP-FITCs may continue to move into cells due to the magneticforce and may have been engulfed by the cells themselves (FIG. 30B). TheFACS results shows that standing for two hours on the magnet is enoughfor FITC delivery into cells driven by magnetic forces because noidentifiable difference is observed for standing on the magnet for 2, 4and 6 hours (FIG. 28A, B). The FITC delivery efficiency is about 100%for standing for 2, 4 and 6 hours. FIG. 28C shows no cytotoxicity formGNPs for both 24 and 48 hours among concentrations ranging from 4.7-75nmol Au mL⁻¹ using the MTS method.

In order to confirm the results of the FITC delivery into the KG-1 cellline, images from both fluorescent and confocal microscopy are taken(FIG. 29). Compared with the blue channel (checking cell nucleus), theimage (FIG. 29A) in the green channel (fluorescent signal) offluorescent microscopy showed that not all KG-1 cells took up themGNP-FITCs even though the FITC delivery efficiency is about 100%according to FACS results. This error may have arisen due to thelimitations of the analytic methods of the FACS instrument. According tothe confocal image in FIG. 29B, we can clearly see that the greenfluorescent signal surrounded the nucleus of the cells, there are someespecially highlighted spots near the nucleus, which confirmed thatmGNP-FITCs actually entered into KG-1 cells and migrated towards cellnucleus.

Sonication can disperse iron oxide nanoparticles into smallernanoparticles and also make gold cations adsorb on the surface or becometrapped in the micropores of the iron oxide nanoparticles. Through aquick reduction of ascorbic acid and post-HCl solution treatment, mGNPswith a uniform spherical morphology and sizes around 20-30 nm can besynthesized in a water solution. The mGNPs have a core-shell structure.mGNPs are non-cytotoxic and mGNP-FITCs can enter into the KG-1 cellline, which is confirmed by the confocal images.

Example 12 FITC Delivery into Plant Cells with and without Cell WallsUsing Magnetic Gold Nanoparticles

Cell culture: MD cell suspensions of canola (B. napus L. var. Jet Neuf)are maintained on a rotary shaker (160 rpm) at 20° C. in NLN media(pH6.0, containing 6.5% sucrose, 30 mg/L glutathione, 800 mg/Lglutamine, 100 mg/L L serine, 0.5 mg/L a-naphthaleneacetic acid (NAA),0.05 mg/L 6-benzylaminopurine (BA) and 0.5 mg/L 2,4-D). At 2-weekintervals, one third of the mass of cells grown in 125 mL flasks istransferred to 50 mL of fresh NLN medium. Seeds of carrot (D. carota L.var. Konservnaja 63) are obtained from Plant Gene Resources of Canada(Saskatoon, Saskatchewan). Cells derived from leaves of in vitro plantsare cultured in MS media, 3% sucrose, 0.2 mg/L BA, 1.0 mg/L NAA (pH=6).Two to Three days after subculture, cells are used for protoplastisolation.

Protoplast isolation: Plant cells are preplasmolyzed by incubation inCPW13M solution for 1 hour at room temperature. The solution is thenreplaced with a digestion solution, consisting of ½ MS salts, 0.06%2-(N-Morpholino)ethanesulfonic acid (MES), 13% mannitol, 0.1% MacerozymeR-10 (Yakult Honsha Co., Japan) and 0.5% Cellulase Onozuka R-10 (YakultHonsha Co., Japan), pH 5.8. The incubation is carried out overnight (16hrs) at 25° C. in the dark. The digestion mixture is filtered through asterile nylon cell strainer (40 μm, BD Falcon, USA) to remove thedebris, and then centrifuged (100×g) for 10 min. The pellet isresuspended in CPW255 and 2 mL of CPW13M is added to the top. Theprotoplasts are then collected with sterilized Pasteur pipettesfollowing centrifugation (100×g) for 10 min, washed twice, and finallyresuspended in ½ NLN medium supplemented with 13% mannitol. Theprotoplast solution is used for the mGNP-FITC delivery experiment.

Synthesis of mGNP-FITC: (1) 0.0116 g HS-PEG-NH₂ (MW 5000) is dissolvedinto a 20 mL vial containing 10 mL of MilliQ water. 1 mL of the preparedmGNP solution is transferred into this vial and stirred for 5 min. Thevial is kept at 4° C. overnight. (2) The solution is centrifuged at10000 rpm for 30 min. The supernatant is discarded and the sediment iswashed once using the same centrifuge conditions. The sediment isdissolved in 0.5 mL of MilliQ water (mGNP solution). Meanwhile, 100 mgFITC is dissolved into 0.5 mL DMF, then mixed with the above mGNPsolution. The mixture is stirred for 5 minutes, then kept in roomtemperature overnight. The mixture is dialyzed until there is no freeFITC in solution.

Magnetic-field-driven cellular uptake experiment: Protoplasts with adensity of 5×10⁵ cells/plate are placed in 35 mm culture dishes, thedishes are sealed with parafilm. The magnetic-field-driven deliverymethod is carried out by placing the culture dishes containing 1 mL ofmedium with 0.25 μg/mL mGNP-FITC or mGNP on the top of an Nd—Fe—Bpermanent magnet for 12 hrs. The protoplasts are then collected, fixedin 2% paraformaldehyde and completely washed twice with PBS and 70%ethanol, respectively.

Cell viability: Protoplasts are seeded in 35 mm Petri dishes in culturemedium. 30 μL of mGNPs is added into each dish. The Petri dishes are puton top of the magnet at room temperature overnight. A drop of cellsolution is deposited on a microscope glass slide and stained with FDA.Images are taken with both bright and green channel under a fluorescentmicroscope (Leica CW 225 A with Nikon digital camera DXM1200). Theprotoplast numbers are counted under bright channel and fluorescentchannel. The cell viability or NPs cytotoxicity is then calculated.

Flow Cytometry Measurement: Protoplasts exposed to mGNP-FITC atdifferent concentrations are collected and centrifuged at 1000 rpm for10 min. The collected cells are extensively washed using PBS then fixedin 2% paraformaldehyde and resuspended in 400 μL PBS. The mGNP-FITCdelivery efficiency is evaluated with Flow Cytometry (FACscan,Becton-Dickinson, San Jose, Calif., USA) at an excitation wavelength of488 nm.

Atomic force microscope (AFM) image: A small amount of sample solutionis directly transferred dropwise onto the silicon wafer. The sample iscovered and kept at room temperature until the solution is dry. AFMimages are taken using Veeco Multimode V SPM operating in tapping mode.

Confocal microscopy imaging of plant cells: Protoplasts are seeded at adensity of 1×10⁵ cells/cm² on cover slips previously coated withpoly-L-lysine (10 μg/mL) for 45 min. The protoplasts are exposed to 0.25μg/mL mGNP-FITC and mGNP (the control) on an Nd—Fe—B permanent magnet.Uptake is terminated by washing the cells twice with PBS buffer andtwice with 70% ethanol, separately. After 12 hours of incubation on anNd—Fe—B permanent magnet, the cells are fixed in 2% paraformaldehyde andexamined under a confocal laser scanning microscope (Quorum WaveFX-Sinning Disk) equipped with imaging software—Hamamatsu EMCCD(C9100-13).

TEM image: TEM images are taken using a Philips-FEI Morgagni 268instrument, operated at 80 kV. The sample solution is deposited on thecopper support, which is coated with carbon. Protoplasts are fixed in 2%glutaraldehyde in 4% PEN cacodylate buffer, pH 7.2, for 2 hours at roomtemperature. (a) The fixative solution is drained off and replaced with0.1M PBS buffer. Two further changes are done 10 minutes apart. (b) Thebuffer is drained and the sample is post-fixed with 1% osmium tetroxide(OSO₄ in 0.12 M Cacodylate buffer, pH 7.2) for one hour. (c) The sampleis washed using 0.1M phosphate buffer 3 times for a total of one halfhour. (d) The sample is dehydrated through a graded ethanol series asfollows: 50%, 70%, 90%, 100×3 changes; one change every 15 minutes. (e)The ethanol is drained from specimen and new ethanol: Spurr mix is addedfor 3 hours. The ethanol: Spurr mix is replaced with pure Spurr resin.The Petri dish is sealed overnight. (f) The Spurr resin is replacedagain and the sample is dried at 70-80° C. in an oven for 18 hours. (g)The sample is cooled and then removed from molds. (h) The sample isultracut by Reichert-Jung Ultramicrotome and stained with uranyl acetateand lead citrate.

Results and Discussion

Synthesis of mGNP-FITC

Core-shell mGNPs are used to covalently bind FITC (FIG. 31). The mGNPshas a spherical morphology and are about 20-30 nm in size. Thecore-shell of the mGNPs is made of an iron oxide core covered completelyby gold (15). When mGNPs are reacted with thiol PEG-NH₂ (MW 5000), thethiol functional groups interacted with gold while the amino groupserved as a free functional groups. Due to the spherical structure ofmGNP, the amino groups distributed evenly around mGNP like a ball. AfterFITC reacted with amino groups, the FITC spherically mounted on themGNP's surface as shown in FIG. 31.

According to the FIG. 36, it can be seen that most cells in brightchannel appeared in the green channel as well, which shows that mostcells had green fluorescent signals as well. This result is consistentwith our FACS results (FIG. 32).

FITC Delivery into Protoplasts (Plant Cells without Cell Wall)

FACS results in FIG. 32 show that the FITC delivery efficiency is about100% with mGNPFITC concentrations from 4.7 to 18.8 nmol Au/mL. Forcanola protoplasts, the difference of fluorescent strength among thethree concentrations is very small (FIG. 32-Canola protoplast A), butthe fluorescent strength for carrot protoplasts is quite different (FIG.32-Carrot protoplast A). The stronger fluorescent signals reflect thehigher FITC concentration. This phenomenon is caused by the differentprotoplast size: the size of canola protoplast is about three-timeslarger than that of carrot protoplast (FIG. 36). The images offluorescent microscopy in FIG. 36 also supports our FACS results. Thegreen fluorescent signals appears in most canola and carrot protoplasts,showing that most of mGNP-FITCs enters protoplasts. The cell countdistributive curves in the control cells are different from that ofnormal mammalian cell lines. The fluorescent strength of normalmammalian cell lines is around 10⁰-10¹, but the fluorescent strengths ofthese two protoplasts are at 10⁰-10², which indicates that somefluorescent signals is coming from the protoplasts themselves. Theseresults are thought to come from the remaining cell wall (the enzymaticremoval of cell walls is not 100% efficient and some cell walls stillremains) as some fluorescent signals from cell wall of canola cellsunder the fluorescent microscopy may still be observed. Even though somecell wall remains, the distributive curves of cell counts is shiftedoverall to the position indicating stronger fluorescent strength forboth canola and carrot protoplasts after delivery using mGNP-FITC. Itseems that mGNP-FITC enters into walled plant cells. FIG. 32B shows thatmGNPs have no cytotoxicity because the cell viability after contactingwith mGNPs is similar to that of the control cells.

According to the confocal images (FIG. 33), the FITC completely enteresboth canola and carrot protoplasts. Compared with control cells, strongfluorescent signals appears near the blue nuclei. For canolaprotoplasts, there are several small spherical green signals surroundingthe blue nuclei. This result is consistent with the FITC's sphericaldistribution surrounding mGNPs (shown in FIG. 31). When an mGNP enters acell, it carries all FITCs bound on the surface of mGNP, and thus showsthe spherical morphology. The different mGNPs inside the cellconstitutes different green fluorescent spheres. Therefore, severalmGNPs must have entered the cell. The size difference in the sphericalfluorescent signal is caused by the aggregation of mGNPs or differentdistances near the confocal section. For carrot protoplasts, thisphenomenon cannot be clearly seen due to their smaller size. In order tosupport our hypothesis, sectional TEM imaging is also performed. A largenumber of mGNPs inside cells is observed. FIG. 3 shows that mGNPs existsnot only inside (FIG. 34-Canola C and Carrot B) and outside the endosome(FIG. 34-Canola B, C and Carrot C) but also inside nucleus (FIG.34-Canola A and Carrot B). According to the size analysis, most mGNPsaggregated in organelles. Because mGNPs are covered by PEG, they arestable and do not aggregate. The aggregated mGNPs show that the chemicalbonds between gold and thiol functional groups may have been brokenafter the mGNPs entered cells. These results also show that mGNPs cancarry biomolecules into cell nuclei, which provides a new method todeliver genes into plant cells because usually only genes which enterthe nucleus can be expressed. FIG. 34-Carrot A shows that when an mGNPenters into the cell, an endosome is formed. Therefore, it can behypothesize that mGNPs may enter cells through an endocytotic process.The mGNPs first enter into an endosome, then enter other organelles.

For walled canola cells, it is found that some mGNPs went through cellwall according to the sectional TEM images (FIG. 35). In FIG. 35A, amGNP just entered cell, near the cell wall, three mGNPs are in thecytoplasm and two mGNPs are in endosomes. In FIG. 35B, two mGNPs aregoing through the cell wall. According to the size and contrastanalysis, these two mGNPs are carrying PEG molecules. The pictureconfirmed the chemical bond between FITC and mGNP did not break when themGNP-FITC penetrated the cell wall. In FIG. 35C, two mGNPs are goingthrough the cell wall. From the zoomed-in image (FIG. 35D), it isclearly seen that an mGNP is going through the cell wall but stoppednear inside the cell wall. Combined with the results in FIG. 34, it canbe concluded that, when mGNPs entered cells, the FITC remains bound withthe mGNPs. When mGNPs enter other organelles, the FITC and PEG moleculesmay be decomposed by the enzymes and separated from the mGNPs, with thenanoparticles left in the organelles, which results in mGNP aggregationonce nanoparticles became unstable.

mGNPs with uniform size and spherical morphology are covalently bondedwith FITC, and are delivered into plant cells with and without cellwalls driven by an external magnetic force. Two types of plant cells,canola and carrot cells, are tested. The FITC delivery efficiency isabout 100% for both protoplasts according to FACS results. These resultsare also confirmed by the confocal and sectional TEM images. Accordingto the sectional TEM images, mGNPs distributed in endosomes, the nucleusand the cytoplasm of canola and carrot protoplasts, but most mGNPsaggregated in organelles. The sectional TEM images also confirm thatmGNPs does pass through the cell walls of canola cells, which indicatedthe mGNPs have the ability to directly enter walled plant cells, whichis very important for plant transformation.

REFERENCES

The following references are representative of the level of skill in theart and are incorporated herein as if reproduced in their entirety(where permitted).

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While preferred embodiments have been described above and illustrated inthe accompanying drawings, it will be evident to those skilled in theart that modifications may be made without departing from thisdisclosure. Such modifications are considered as possible variantscomprised in the scope of the disclosure.

1. A method of delivering a molecule across a cell membrane using adelivery vehicle comprising a molecule fixed on a magnetic nanoparticle,the method comprising the steps of: (a) positioning the nanoparticle inthe immediate vicinity of the cell membrane; and (b) simultaneouslysubjecting the nanoparticle and cell membrane to magnetic field andultrasound.
 2. The method of claim 1 wherein the ultrasound compriseslow-intensity pulsed ultrasound.
 3. The method of claim 1 wherein thenanoparticle comprises a single-walled carbon nanotube.
 4. The method ofclaim 1 wherein the nanoparticle comprises a biodegradable orbiocompatible material.
 5. The method of claim 4 wherein thenanoparticle comprises silica.
 6. The method of claim 1 wherein themolecule comprises a DNA or a RNA molecule.
 7. The method of claim 1which is practiced in vivo, and wherein the delivery vehicle areconcentrated in a specific region by a magnetic force placed adjacentthe specific region, and forced across a cell membrane by a magneticfield.
 8. The method of claim 7 wherein the magnetic field alternatesdirection across the specific region after concentration of the deliveryvehicle.
 9. The method of claim 7, wherein the specific region is behindor associated with the blood-brain barrier.
 10. The method of claim 1,wherein said membrane is from a cell chosen from a mammalian cell and aplant cell.
 11. The method of claim 10, wherein said mammalian cell ischosen from a normal cell or a cancer cell.
 12. The method of claim 10,wherein said plant cell further comprises a cell wall.
 13. The method ofclaim 10, wherein said plant cell is chosen from a canola cell or acarrot cell.
 14. The method of claim 12, wherein said plant cell ischosen from a canola cell or a carrot cell.
 15. The method of claim 11,wherein said cancer cell is chosen from a MCF-7 cell, a HeLa cell, aKG-1 cell, a breast cancer cell, a cervix cancer cell, and a human acuteleukemia cell.
 16. The method of claim 1, wherein said magneticnanoparticle is chosen from a magnetic gold nanoparticle (mGNP), amagnetic single wall carbon nanotube (mSWCNT), or combinations thereof.